A deficiency in aqueous humor outflow through the trabecular meshwork (TM) underlies the increased intraocular pressure (IOP) found in primary open angle glaucoma (POAG), and is associated with an accelerated loss of endothelial cells from the trabecular beams. Loss of meshwork cellularity has been postulated to play a role in allowing or promoting the fusion of trabecular beams and the degradation of meshwork structure that is thought to contribute to decreased facility in POAG. Understanding the causes of accelerated cellular loss is therefore crucial to understanding the etiology of this disease. We have studied primary explant cultures of living POAG TM cells using real time, high resolution video-enhanced differential interference contrast (VE-DIC) light microscopy. Preliminary studies found striking differences in intracellular vesicle movement and organization (microtubule-based functions), and filopdial formation and membrane integrity (actin-based functions) compared with control cultures. In living eyes, microtubule defects would alter the ability of TM cells to divide, and in non-dividing cells would interfere with sorting and transport of intracellular constituents to the cell periphery. Defects in actin utilization would degrade plasma membrane tone, alter phagocytosis, and decrease the ability of TM cells to crawl or contract. Our hypothesis is that the TM cells in POAG are defective in microtubule and actin based cytoskeletal function, and that this contributes to TM cell loss and trabecular meshwork degeneration in vivo. We will use high magnification VE-DIC microscopy together with frame-by-frame analysis to determine whether there is a defect in vesicle and organelle movement or organization in first generation explant cultures of POAG and control TM cells. These cultures will also be used to determine whether there are fewer microtubules or less dynamic microtubules in POAG cells, and whether there are more aberrant filopodia changes in plasma membrane tension, or changes in F-actin structure. Understanding the ways in which the cytoskeletal systems are defective in POAG TM cells will point to new targets for pharmacological intervention, and provide a rational basis for future therapies, such as genetic manipulations, that are directed at restoring normal function to trabecular meshwork cells in vivo.